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Golgi-like staining with haematoxylin
Neuroscience Letters, 18 (1980) 31-36
31
© Elsevier/North-Holland Scientific Publishers Ltd.
GOLGI-LIKE STAINING WITH HAEMATOXYLIN
PETER G.H. CLARKE lnstitut d'Anatomie, Universit~ de Lausanne, Rue du Bugnon 9, 1011 Lausanne-CHUV (Switzerland)
(Received January 28th, 1980) (Accepted February 28th, 1980)
SUMMARY
Under certain conditions the Loyez method [4], an iron-haematoxylin stain for myelin, will impregnate the perikarya, dendrites and axons of neurones. This occurs (in chick embryos and young mice) at certain stages of normal development, and may be provoked in adult animals by a lesion of the brain; but it does not occur in normal, unoperated adults.
The phenomenon discussed was first noticed in a series of electrophysiological experiments, in collaboration with Professor Whitteridge at Oxford [3], on the adult cat's visual cortex. Electrolytic lesions were made to m a r k the recording sites. The animals were fixed by perfusion with buffered formalin (pH 7.4), celloidin sections were prepared, and some of these were treated with the Loyez method [4], which is known to stain myelin, erythrocytes and nucleoli. I found that, in addition to these elements, which were stained ubiquitously, m a n y neurones around the lesions were impregnated, including m a n y of their processes (Fig. 1). However, this occurred only if the lesion had been made less than about 5 h before fixation. After longer periods only debris was stained, suggesting that the affected neurones had probably disintegrated. Observations on adjacent Nissl-stained sections were compatible with this interpretation; for after survival times of less than about 5 h relatively heavily Nissl-stained neurones surrounded the lesions, but after longer periods no neurones were detectable at corresponding sites. T h e possibility that the staining involved some particular interaction with the metal (tungsten) of the electrode was tested on the cerebral cortex of a rat, using electrodes made of tungsten, steel or platinum, and passing either positive or negative current (10-20 ~A for 10-20 sec). For every combination of electrode metal and current direction, there was a clearly demarcated patch around the lesion, of radius about 150 #m, in which about half the neurones were stained. Apart from very occasional impregnated neurones
32
Fig. 1. Top: low-power view of a Loyez-stained section through the edge of an electrolytic lesion in layer I1 of area 17 in an adult cat, showing that neurones are impregnated exclusively around the lesion. The core of the lesion is out of the section. Lower left: medium-power view of the region of stained cells in the same section. Lower centre and right: tracing and photographic montage of a Loyez-stained pyramidal cell in an adjacent section through the same lesion. The perikaryon lies at the border between layers 1I and 11I. The sections are sagittal, and approximately perpendicular to the cortex. Scales: top, 200 0.m; lower left, 40 #m; right, 25 ,am; allowing for measured shrinkage to 70%.
33 elsewhere along the electrode track, no other neurones were impregnated. This all implies that the neurones were stained simply because they were damaged or degenerating. This suggested that the Loyez method might demonstrate selectively the many neurones which degenerate during normal development (e.g. refs. 1, 2 and 5), and so it was tried on some 32 chick embryos of various ages. Brains were fixed with 1007o formalin in 0.1 M phosphate buffer (pH 5.8-7.5), embedded in celloidin, and sectioned at 30-60/~m. The sections were stored in 70% ethyl alcohol, and subsequently stained by a procedure which is essentially that of Loyez [4]. They were rinsed for 5 min in distilled water, mordanted for 24 h in a 407o aqueous solution of ferric a m m o n i u m sulphate, and then rinsed for 5 min in tap water before being transferred to the haematoxylin solution, which was made up from a ripened stock of 10% haematoxylin in absolute ethyl alcohol. The stock was diluted 10-fold with distilled water, and 207o by volume of saturated lithium carbonate solution was added. After 12-24 h in the haematoxylin bath, the sections were rinsed thoroughly in tap water (two changes of 5 min each), and then differentiated in Weigert's differentiator (2o7o (w/v) borax, 2.507o (w/v) potassium ferricyanide, in distilled water). The time for differentiation was critical, and had to be controlled under the microscope; the optimum was usually between 1 and 2 min. Finally, the sections were washed in distilled water, dehydrated in ethanols, cleared in xylene, mounted on slides and coverslipped. Several variations have been tested, using sections from the brains of 14-day chick embryos, and so far the above procedure impregnates neurones and their processes the most reliably and completely. With other fixatives - glutaraldehyde, acrolein, chloral hydrate, ethyl alcohol, Carnoy's medium, or even acid formaldehyde (pH _<5) - the neurones were not stained; with acetaldehyde, only a very few were stained. Ferric a m m o n i u m sulphate is acceptable as an alternative differentiator, but 0.1 070 hydrochloric acid in 70°7o ethyl alcohol leads to poorer neuronal staining. In paraffin and frozen sections, staining was weak and restricted to a perikaryon of a few neurones, including some that were consistently not impregnated following celloidin embedding. During the last half of the incubation period, numerous neurones were impregnated, including often, but not always, their dendrites and axons (Fig. 2). In impregnated cells that were not completely opaque, one could usually discern that the plasma membrane was relatively clear, but that there were one or several dark clumps within the cell. The overall pattern was roughly predictable according to age; some cell groups were virtually never stained, but in others m a n y neurones were stainable over a specific period lasting 5-1 1 days depending on the cell group. The largest neurones generally stained most readily and completely. In some structures there was a gradient of preference for the stain; for example, throughout the last 10 days of incubation, the stratum griseum centrale of the tectum usually contained numerous heavily impregnated neurones in its ventromedial region, less
Fig. 2. Loyez-stained neurones in coronal sections of normal, unoperated chick embryos. Top: montage of a large neurone located exactly on the midline in the brain stem of a 14-day-old embryo. The arrow indicates the neurone's axon. Middle: low power view oF the same e mbryo' s brian stem, at the level of the Vllth nerve nucleus. The large scattered neurones (left and bottom) are in the gigantocellular part of the caudal reticulopontine nucleus. Bottom: immature Purkinje cells in an 18-day-old embryo. Scales: 50 "am top; 300 ,am middle and 100 ,am bottom, allowing for shrinkage to 70%.
35 dorsomedially, and fewest dorsolaterally. In embryos fixed during the last day or so of incubation, the impregnation was less complete in most structures; dendrites stained weakly if at all at this stage, and the impregnation of the soma was frequently incomplete, in m a n y cases being largely restricted to the nucleus. Two days after hatching, impregnated neurones were rare. I have tried the Loyez method also on 1- and 3-day-old mice, and groups of impregnated neurones occurred as in the chick embryos. The pattern described above is not what one would expect if the Loyez method selectively demonstrates dying neurones. Indeed, careful comparison with the published data on cell death in the chick (e.g. refs. 1,2 and 5) indicates that m a n y of the stained cells cannot be dying, and many dying cells stain only very briefly, if at all. For example, a variable number of neurons, but sometimes as many as 3 0 - 4 0 % , may be impregnated in the medial and lateral pontine nuclei towards the end of incubation, yet there appears to be no cell death in these nuclei during development [1]. Conversely, very few (never more than 10) neurones are stained in the whole of the isthmo-optic nucleus during the period between 13 and 16 days of incubation, when more than half its 22,000 neurones are known to die [2]. Again, staining in the mesencephalic Vth nucleus is entirely restricted to its lateral division and continues almost to the time of hatching, yet the massive 75°70 cell loss which occurs in this nucleus has been shown to involve both the medial and the lateral divisions, and to be complete by embryonic day 13 [5]. Zaprianova [6] has noticed that Baker's acid haematein method stains certain neurones in chick embryos, but very few in adults, and has suggested that the stained material is destined to be transferred from the neurones to developing myelin. Judging from her published results, the Loyez and Baker methods do not necessarily stain the same neurones at the same stages, so the phenomenon that she reported is probably distinct from the present one. Nevertheless, her hypothesis could conceivably be applied to the Loyez-stained neurones. This would not explain, however, why the method is selective for certain groups of neurones, whereas in others - such as the isthmo-optic nucleus and the medial division of the mesencephalic Vth nucleus, which give rise to well myelinated axons - most neurones are never impregnated. The subtances which are Loyez-stained in myelin seem to differ in some sense from those in the neurones, since the myelin stains darkly following fixation with any aldehyde, whereas the neurones require formaldehyde. The present preliminary results may prove useful in at least three ways. (i) They suggest differences in (lipid?) metabolism between the stained and unstained neurones during development. (ii) They provide an additional morphological tool foor the study of developing brains. (iii) They indicate a possible way to find out, in adult brains, which cells have been damaged by a lesion.
36 ACKNOWLEDGEMENTS
I thank Mrs. J.P. Donaldson in Oxford and Mrs. M. Jayet and Mrs. M. Duruz in Lausanne for excellent histological assistance, and Mrs. Ch. Vaclavik for typing the manuscript. I am indebted to my colleagues H. Dubois, D.O. Frost, L.J. Garey, P.P. Giorgi, G.M. Innocenti and H. Van der Loos for many helpful comments on the manuscript. Most of the work was supported by Grant 3.776 to H. Van der Loos from the Swiss National Science Foundation.
REFERENCES 1 2 3 4 5 6
Armstrong, R.C. and Clarke, P.G.H., Neuronal death and the development of the pontine nuclei and inferior olive in the chick, Neuroscience, 4 (1979) 1635-1647. Clarke, P . G . H . , Rogers, L.A. and Cowan, W.M., The time of origin and pattern of survival of neurones in the isthmo-optic nucleus of the chick, J. comp. Neurol., 167 (1976) 125-142. Clarke, P . G . H . and Whitteridge, D., A comparison of stereoscopic mechanisms in cortical visual areas V1 and V2 of the cat, J. Physiol. (Lond.), 272 (1977) 92-93P. Loyez, M., Coloration des fibres nerveuses par la m~thode fl l'hbmatoxyline au fer aprbs inclusion 'fi la celloidine, C.R. Soc. Biol. (Paris), 69 (1910) 511-513. Rogers, L.A. and Cowan, W.M., The development of the mesencephalic nucleus of the trigeminal nerve in the chick, J. comp. Neurol., 147 (1973) 291 320. Zaprianova, E., Histochemistry and morphological metabolism of lipids in the chicken brain in relation to myelination, Acta anat. (Basel), 75 (1970) 276-300.
31
© Elsevier/North-Holland Scientific Publishers Ltd.
GOLGI-LIKE STAINING WITH HAEMATOXYLIN
PETER G.H. CLARKE lnstitut d'Anatomie, Universit~ de Lausanne, Rue du Bugnon 9, 1011 Lausanne-CHUV (Switzerland)
(Received January 28th, 1980) (Accepted February 28th, 1980)
SUMMARY
Under certain conditions the Loyez method [4], an iron-haematoxylin stain for myelin, will impregnate the perikarya, dendrites and axons of neurones. This occurs (in chick embryos and young mice) at certain stages of normal development, and may be provoked in adult animals by a lesion of the brain; but it does not occur in normal, unoperated adults.
The phenomenon discussed was first noticed in a series of electrophysiological experiments, in collaboration with Professor Whitteridge at Oxford [3], on the adult cat's visual cortex. Electrolytic lesions were made to m a r k the recording sites. The animals were fixed by perfusion with buffered formalin (pH 7.4), celloidin sections were prepared, and some of these were treated with the Loyez method [4], which is known to stain myelin, erythrocytes and nucleoli. I found that, in addition to these elements, which were stained ubiquitously, m a n y neurones around the lesions were impregnated, including m a n y of their processes (Fig. 1). However, this occurred only if the lesion had been made less than about 5 h before fixation. After longer periods only debris was stained, suggesting that the affected neurones had probably disintegrated. Observations on adjacent Nissl-stained sections were compatible with this interpretation; for after survival times of less than about 5 h relatively heavily Nissl-stained neurones surrounded the lesions, but after longer periods no neurones were detectable at corresponding sites. T h e possibility that the staining involved some particular interaction with the metal (tungsten) of the electrode was tested on the cerebral cortex of a rat, using electrodes made of tungsten, steel or platinum, and passing either positive or negative current (10-20 ~A for 10-20 sec). For every combination of electrode metal and current direction, there was a clearly demarcated patch around the lesion, of radius about 150 #m, in which about half the neurones were stained. Apart from very occasional impregnated neurones
32
Fig. 1. Top: low-power view of a Loyez-stained section through the edge of an electrolytic lesion in layer I1 of area 17 in an adult cat, showing that neurones are impregnated exclusively around the lesion. The core of the lesion is out of the section. Lower left: medium-power view of the region of stained cells in the same section. Lower centre and right: tracing and photographic montage of a Loyez-stained pyramidal cell in an adjacent section through the same lesion. The perikaryon lies at the border between layers 1I and 11I. The sections are sagittal, and approximately perpendicular to the cortex. Scales: top, 200 0.m; lower left, 40 #m; right, 25 ,am; allowing for measured shrinkage to 70%.
33 elsewhere along the electrode track, no other neurones were impregnated. This all implies that the neurones were stained simply because they were damaged or degenerating. This suggested that the Loyez method might demonstrate selectively the many neurones which degenerate during normal development (e.g. refs. 1, 2 and 5), and so it was tried on some 32 chick embryos of various ages. Brains were fixed with 1007o formalin in 0.1 M phosphate buffer (pH 5.8-7.5), embedded in celloidin, and sectioned at 30-60/~m. The sections were stored in 70% ethyl alcohol, and subsequently stained by a procedure which is essentially that of Loyez [4]. They were rinsed for 5 min in distilled water, mordanted for 24 h in a 407o aqueous solution of ferric a m m o n i u m sulphate, and then rinsed for 5 min in tap water before being transferred to the haematoxylin solution, which was made up from a ripened stock of 10% haematoxylin in absolute ethyl alcohol. The stock was diluted 10-fold with distilled water, and 207o by volume of saturated lithium carbonate solution was added. After 12-24 h in the haematoxylin bath, the sections were rinsed thoroughly in tap water (two changes of 5 min each), and then differentiated in Weigert's differentiator (2o7o (w/v) borax, 2.507o (w/v) potassium ferricyanide, in distilled water). The time for differentiation was critical, and had to be controlled under the microscope; the optimum was usually between 1 and 2 min. Finally, the sections were washed in distilled water, dehydrated in ethanols, cleared in xylene, mounted on slides and coverslipped. Several variations have been tested, using sections from the brains of 14-day chick embryos, and so far the above procedure impregnates neurones and their processes the most reliably and completely. With other fixatives - glutaraldehyde, acrolein, chloral hydrate, ethyl alcohol, Carnoy's medium, or even acid formaldehyde (pH _<5) - the neurones were not stained; with acetaldehyde, only a very few were stained. Ferric a m m o n i u m sulphate is acceptable as an alternative differentiator, but 0.1 070 hydrochloric acid in 70°7o ethyl alcohol leads to poorer neuronal staining. In paraffin and frozen sections, staining was weak and restricted to a perikaryon of a few neurones, including some that were consistently not impregnated following celloidin embedding. During the last half of the incubation period, numerous neurones were impregnated, including often, but not always, their dendrites and axons (Fig. 2). In impregnated cells that were not completely opaque, one could usually discern that the plasma membrane was relatively clear, but that there were one or several dark clumps within the cell. The overall pattern was roughly predictable according to age; some cell groups were virtually never stained, but in others m a n y neurones were stainable over a specific period lasting 5-1 1 days depending on the cell group. The largest neurones generally stained most readily and completely. In some structures there was a gradient of preference for the stain; for example, throughout the last 10 days of incubation, the stratum griseum centrale of the tectum usually contained numerous heavily impregnated neurones in its ventromedial region, less
Fig. 2. Loyez-stained neurones in coronal sections of normal, unoperated chick embryos. Top: montage of a large neurone located exactly on the midline in the brain stem of a 14-day-old embryo. The arrow indicates the neurone's axon. Middle: low power view oF the same e mbryo' s brian stem, at the level of the Vllth nerve nucleus. The large scattered neurones (left and bottom) are in the gigantocellular part of the caudal reticulopontine nucleus. Bottom: immature Purkinje cells in an 18-day-old embryo. Scales: 50 "am top; 300 ,am middle and 100 ,am bottom, allowing for shrinkage to 70%.
35 dorsomedially, and fewest dorsolaterally. In embryos fixed during the last day or so of incubation, the impregnation was less complete in most structures; dendrites stained weakly if at all at this stage, and the impregnation of the soma was frequently incomplete, in m a n y cases being largely restricted to the nucleus. Two days after hatching, impregnated neurones were rare. I have tried the Loyez method also on 1- and 3-day-old mice, and groups of impregnated neurones occurred as in the chick embryos. The pattern described above is not what one would expect if the Loyez method selectively demonstrates dying neurones. Indeed, careful comparison with the published data on cell death in the chick (e.g. refs. 1,2 and 5) indicates that m a n y of the stained cells cannot be dying, and many dying cells stain only very briefly, if at all. For example, a variable number of neurons, but sometimes as many as 3 0 - 4 0 % , may be impregnated in the medial and lateral pontine nuclei towards the end of incubation, yet there appears to be no cell death in these nuclei during development [1]. Conversely, very few (never more than 10) neurones are stained in the whole of the isthmo-optic nucleus during the period between 13 and 16 days of incubation, when more than half its 22,000 neurones are known to die [2]. Again, staining in the mesencephalic Vth nucleus is entirely restricted to its lateral division and continues almost to the time of hatching, yet the massive 75°70 cell loss which occurs in this nucleus has been shown to involve both the medial and the lateral divisions, and to be complete by embryonic day 13 [5]. Zaprianova [6] has noticed that Baker's acid haematein method stains certain neurones in chick embryos, but very few in adults, and has suggested that the stained material is destined to be transferred from the neurones to developing myelin. Judging from her published results, the Loyez and Baker methods do not necessarily stain the same neurones at the same stages, so the phenomenon that she reported is probably distinct from the present one. Nevertheless, her hypothesis could conceivably be applied to the Loyez-stained neurones. This would not explain, however, why the method is selective for certain groups of neurones, whereas in others - such as the isthmo-optic nucleus and the medial division of the mesencephalic Vth nucleus, which give rise to well myelinated axons - most neurones are never impregnated. The subtances which are Loyez-stained in myelin seem to differ in some sense from those in the neurones, since the myelin stains darkly following fixation with any aldehyde, whereas the neurones require formaldehyde. The present preliminary results may prove useful in at least three ways. (i) They suggest differences in (lipid?) metabolism between the stained and unstained neurones during development. (ii) They provide an additional morphological tool foor the study of developing brains. (iii) They indicate a possible way to find out, in adult brains, which cells have been damaged by a lesion.
36 ACKNOWLEDGEMENTS
I thank Mrs. J.P. Donaldson in Oxford and Mrs. M. Jayet and Mrs. M. Duruz in Lausanne for excellent histological assistance, and Mrs. Ch. Vaclavik for typing the manuscript. I am indebted to my colleagues H. Dubois, D.O. Frost, L.J. Garey, P.P. Giorgi, G.M. Innocenti and H. Van der Loos for many helpful comments on the manuscript. Most of the work was supported by Grant 3.776 to H. Van der Loos from the Swiss National Science Foundation.
REFERENCES 1 2 3 4 5 6
Armstrong, R.C. and Clarke, P.G.H., Neuronal death and the development of the pontine nuclei and inferior olive in the chick, Neuroscience, 4 (1979) 1635-1647. Clarke, P . G . H . , Rogers, L.A. and Cowan, W.M., The time of origin and pattern of survival of neurones in the isthmo-optic nucleus of the chick, J. comp. Neurol., 167 (1976) 125-142. Clarke, P . G . H . and Whitteridge, D., A comparison of stereoscopic mechanisms in cortical visual areas V1 and V2 of the cat, J. Physiol. (Lond.), 272 (1977) 92-93P. Loyez, M., Coloration des fibres nerveuses par la m~thode fl l'hbmatoxyline au fer aprbs inclusion 'fi la celloidine, C.R. Soc. Biol. (Paris), 69 (1910) 511-513. Rogers, L.A. and Cowan, W.M., The development of the mesencephalic nucleus of the trigeminal nerve in the chick, J. comp. Neurol., 147 (1973) 291 320. Zaprianova, E., Histochemistry and morphological metabolism of lipids in the chicken brain in relation to myelination, Acta anat. (Basel), 75 (1970) 276-300.